EP1734359A1 - RAMAN spectroscopic analysis method and system therefor - Google Patents

Abstract

A Raman spectroscopy analysis procedure irradiates the sample containing the analyte simultaneously with primary light and medium or far infrared pump light (9) to produce a higher occupation count compared to the thermal equilibrium for at least one vibration or rotation state including an Anti-Stokes line (AS). Independent claims are included for a Raman spectrometer using the procedure.

Description

The invention relates to a Raman spectroscopic analysis method for determining the presence and / or concentration of an analyte in a sample, comprising the steps of: irradiating the sample with narrow band primary light of the near infrared, visible or ultraviolet spectral region, detecting secondary light by interaction the primary light is formed with the sample, recording and evaluating at least one spectral portion of the secondary light containing at least one Raman line, for determining the presence and / or concentration of an analyte in the sample. The invention further relates to a Raman spectrometer for carrying out such a method.

Spectroscopic methods have the great advantage for clinical examinations that, ideally, reagents can be dispensed with and a single sample can be examined simultaneously for different analytes. Despite these advantages, spectroscopic examination methods for the examination of samples obtained from human or animal tissue or body fluids have not been able to prevail over conventional examination methods.

For this reason, reagents are still required in clinical routine for analytical investigations. In this case, the production and storage of the reagents are costly. A further disadvantage of reagent-bound detection methods is that a sample can usually only be examined for a single analyte, which requires the removal of relatively large sample volumes from patients and consequently results in a high load on the patients.

Various approaches to the spectroscopic examination of medical samples are known in the art. For the detection of organic molecules in particular the spectroscopy of molecular vibrations is suitable because vibration states in infrared and Raman spectra can be identified by characteristic lines and assigned to analyte molecules. A summary of various methods can be found in the article by W. Petrich, "Mid-infrared and Raman spectroscopy for medical diagnostics", Appl. Spectrosc. Rev. 36 (2001), 181-237 ,

Raman spectroscopic studies make use of the fact that light of the near infrared, visible or ultraviolet spectral region of molecules is inelastically scattered to a small extent. Typically, this inelastically scattered part accounts for only about one millionth of the incident light intensity.

In an inelastic scattering process, energy is exchanged between scattering molecules and incident primary light. The scattering molecule can absorb energy and pass into an excited state of vibration or release vibration energy during the scattering process and into a lower vibration state (usually in the ground state). In this process, called Raman scattering, the wavenumber of the scattered secondary light relative to the incident primary light changes by values corresponding to the difference in energy of vibrational states of the scattering molecule. For organic molecules, such as glucose, the energy gap between adjacent vibrational states is typically about 0.12 eV, which corresponds to a wavenumber of about 1,000 cm ^-1 in spectroscopic studies. An example is the COH bending vibration of the glucose molecule with a wavenumber of 1077 cm ^-1 .

In the Raman spectrum of the secondary light, which results from the interaction of the primary light with the sample, so-called Raman lines are found next to the line of the incident primary light whose wavenumbers are dependent on the wavenumber of the incident primary light as a function of the energy distances of adjacent vibrating states of the scattering Differentiate molecules. In a Raman spectrum, red-shifted Raman lines are called Stokes lines with respect to the primary light and Raman lines that are blue-shifted with respect to the primary light are called anti-Stokes lines.

When examining samples obtained from human or animal tissue, body fluids or respiratory gases, the Raman lines typically have wavenumbers that deviate from the wavenumber of the primary light by about 1000 cm ^-1 , the ratio of the light intensity of the Raman lines many orders of magnitude below the light intensity of the incident primary light.

Based on the distances and intensities of the Raman lines from the line of incident primary light, analytes in a sample can be identified. The intensity of the Raman lines is proportional to the analyte concentration, so that with Raman spectroscopy, a determination of analyte concentrations is possible.

However, analyte concentrations below about 0.1 mmol per liter can not be detected by known spectroscopic assay methods, as in Rohleder et al., "Comparison of mid-infrared and Raman spectroscopy in the quantitative analysis of serum", J. Biomed. Opt. 10 (Issue 3/2005, in press ) for the case of human serum. This limited detection sensitivity is a major reason why spectroscopic methods have not been successful in clinical laboratory diagnostics so far.

The object of the invention is therefore to show a way in which the detection sensitivity of spectroscopic examinations of samples of human or animal tissue, body fluids or gases can be increased.

This object is achieved by a method of the type mentioned in the present invention that the sample for exciting vibration and / or rotational states of the analyte contained in the sample is irradiated with pump light of the central or far infrared spectral range, so that upon interaction of the primary light with the sample for at least one vibrational and / or rotational state of the Anlayten contained in the sample is present in relation to the thermal equilibrium increased occupation number.

This object is further achieved by a spectrometer comprising a pump light source for irradiating the sample. Pumplicht the middle or far infrared spectral range comprises.

This surprisingly simple measure can increase the signal-to-noise or signal-to-background ratio in the evaluation of anti-Stokes lines by more than an order of magnitude so that analytes in samples obtained from human or animal tissue or body fluids were detected with a detection sensitivity that could not even be achieved with spectroscopic examination methods. For example, without the use of reagents, carbohydrate, lipid (both high density HD and low density LD) and protein concentrations as well as glucose, cholesterol or triglyceride concentrations in medical samples can be determined.

The inventive method is not limited to Raman spectroscopic investigations, so that instead of Raman lines and other spectral properties of the secondary light, such as fluorescent light, can be evaluated. However, it is important that the irradiation of the pump light causes the rotational and / or vibrational states of the analyte contained in the sample to be excited. This means that the energy gaps between the rotational and / or vibrational states, by means of which the analyte is to be detected, are sufficiently large, so that the respective ground state has a significantly higher occupation number than an overlying first excited state.

Namely, both the ground state and the respective first excited rotation or vibration state occupied to the same extent, so can be achieved by irradiation of pump light no change in the occupation and consequently no change in the signal-to-noise or signal-to-background ratio.

Sufficient energy gaps exist between rotational and vibrational states excited by infrared radiation of the mid or far infrared spectral range. For this reason, pumping light of the middle or far infrared spectral range is used in the method according to the invention. In the context of the application, the spectral range from 2 μm to 25 μm is meant by the mid-infrared spectral range (MIR) and the spectral range from 25 μm to 1000 μm by the far-infrared spectral range (FIR), the wavelength data referring in each case to air.

In particular, when using pump light of the far infrared spectral range, the sample can be cooled, so that there are larger differences in the thermal equilibrium between the occupation numbers of the first excited vibration or rotation state and the associated ground state. Upon irradiation of pumping light, the occupation rate of the first excited state, which is even greater than that of the thermal equilibrium, results.

The method according to the invention is not only suitable for the examination of samples obtained from human or animal tissue or body fluids, but can also be used for the examination of other samples. The method according to the invention is also particularly well suited for the investigation of gaseous samples, for example for the analysis of combustion gases or the expelled breath of humans or animals. An advantage of the method according to the invention is in particular that an investigation without destruction of the sample, in particular without destruction of the analyte molecules, is possible.

Fig. 1

eine prinzipielle Darstellung des Raman-Prozesses anhand eines Termschemas von Vibrationszuständen eines Analytmoleküls bei thermischer Vibrationsanregung.

Fig. 2

eine Darstellung gemäß Fig. 1 bei erfindungsgemäßer Anregung der Vibrationszustände durch Einstrahlen von Pumplicht des mittleren infraroten Spektralbereichs.

Fig. 3

eine schematische Darstellung der Intensitäts-verhältnisse von Raman-Linien bei thermischer Anregung der Vibrationszustände und zum Vergleich bei erfindungsgemäßer Anregung durch Einstrahlen von Pumplicht.

Fig. 4

eine schematische Darstellung eines erfindungsgemäßen Raman-Spektrometers,

Fig. 5

ein weiteres Ausführungsbeispiel eines erfindungsgemäßen Raman-Spektrometers.

Further details and advantages of the invention will be explained below with reference to an embodiment of a Raman spectrometer according to the invention with reference to the accompanying figures. The features set forth herein may be used alone or in combination to provide preferred embodiments of the invention. Show it:

Fig. 1

a schematic representation of the Raman process using a Termschemas of vibration states of an analyte molecule during thermal vibration excitation.

Fig. 2

a representation according to FIG. 1 in accordance with the invention excitation of the vibration states by irradiation of pumping light of the central infrared spectral range.

Fig. 3

a schematic representation of the intensity ratios of Raman lines in thermal excitation of the vibration states and for comparison in accordance with the invention excitation by irradiation of pump light.

Fig. 4

a schematic representation of a Raman spectrometer according to the invention,

Fig. 5

a further embodiment of a Raman spectrometer according to the invention.

FIG. 1 shows a greatly simplified term scheme of vibration states of an electronic ground state of an organic molecule. The individual vibration states ν are characterized by horizontal lines represented and designated by numbers 1 to 4. The vibrational ground state is denoted by ν = 1, excited vibrational states with correspondingly higher numbers.

The greater the energy of a vibration state, the further above is shown in Fig. 1, the corresponding line. For simplicity, no rotational states are shown in FIG. In principle, rotational states can be used in an analogous manner as vibrational states for the spectroscopic determination of the presence and / or concentration of an analyte and excited by irradiation of pumping light.

Quantum mechanically, the Raman scattering can be understood so that an incident photon of the primary light is first absorbed by the scattering molecule. Thereby, the molecule is excited from its current vibration state (e.g., ν = 1) to a virtual state, represented by a dotted horizontal line in FIG. The excitation process associated with the absorption of a photon is shown in FIG. 1 by an arrow pointing upwards. With elastic scattering, the molecule changes from this virtual state to its original vibration state by emitting a photon (not shown). In the case of the Raman scattering shown in Fig. 1 by a downward arrow, the molecule is not in its original but a different vibrational state, so that the wavenumber of the emitted Sekundärlichtphotons of the wavenumber of the absorbed Primärlichtphotons by the difference Vibration energy of the involved vibration states different.

At the far left in Fig. 1, the case is shown that the molecule originally in its vibrational ground state (ν = 1), enters a virtual state by absorption of a primary light photon, and then changes to an excited vibration state (v = 2). In this process, the energy of the emitted secondary light photon is smaller than the energy of the absorbed primary light photon by the difference of the vibrational energy of the two vibration states ν = 1 and ν = 2. The secondary light photon is thus red-shifted relative to the primary light and the scattering process shown belongs to a Stokes line, for which reason the relevant arrow is designated by the letter S.

If the molecule is in an excited vibration state upon absorption of the primary light photon (eg, ν = 2 or ν = 3, see Fig. 1, center and right), it can change from a virtual state by emission of a secondary light photon either to an energetically higher vibrational state (v = 3 or ν = 4) or an energetically lower vibrational state (v = 1 or ν = 2).

In Fig. 1, downwardly directed arrows shorter than the associated upwardly directed arrow, labeled with the letter S and downwardly directed arrows longer than the associated upwardly directed arrow, are labeled AS to indicate that the illustrated scattering processes are too high a Stokes line (S) or to an anti-Stokes line (AS).

The intensity of the different Raman lines is related to the number of available molecules in the respective vibration states. At room temperature, most of the molecules are in their vibratory ground state ν = 1. Only a small fraction of the molecules vibrate in a state of vibration. In Fig. 1 is the relatively strong occupation of the vibration ground state ν = 1 by a large circle and the much smaller occupation of the excited vibration state ν = 2 indicated by a much smaller circle. The much weaker occupation of the vibration state ν = 3 is shown without a circle.

For thermodynamic reasons, the occupation of the vibration states is determined by the Boltzmann factor. The ratio between the intensity of an anti-Stokes line I _AS and the intensity of an associated Stokes line I _S is proportional to the factor exp (-ΔE _vib / kT). ΔE _{vib is} the energy difference between the involved vibration states, k the Boltzmann constant and T the temperature. For typical values of ΔE _vib = 0.12 eV, it follows that at room temperature the intensity of the anti-Stokes line is only about 0.8% of the intensity of the associated Stokes line. Because of their significantly greater intensity, prior art Stokes lines are therefore preferred over anti-Stokes lines for assaying samples.

Raman spectroscopic examinations of samples obtained from human or animal tissue or body fluids are complicated by the appearance of fluorescent light. Biological samples often show strong fluorescence upon exposure to primary light. In the context of the invention, it has been recognized that the evaluation of the anti-Stokes lines by fluorescence light is only insignificantly impaired, since fluorescence light is shifted red relative to the incident primary light. Nevertheless, the significantly weaker intensity of anti-Stokes lines results in a poorer signal-to-noise ratio overall and thus a worse one under thermal vibration excitation Detection sensitivity as in an evaluation of Stokes lines according to the prior art.

In the present invention, it has been recognized in this connection that analyte molecules can be brought from the basic vibration state into the first excited state of vibration by irradiating the sample with pump light of the middle infrared spectral region. The occupation of the vibration states is then no longer determined by the Boltzmann factor, but by the interaction of the analyte molecules with the radiated pump light.

In this way, the intensity of the anti-Stokes lines can be significantly increased, since this is proportional to the number of molecules in excited vibration states. If the pump light has a sufficient intensity, then it can be achieved in the ideal case that in the first excited vibration state ν = 2 there are almost as many analyte molecules as in the vibratory ground state ν = 1. By means of methods of population transfer, in principle, even a population inversion can be established achieve an even greater increase in the intensity of anti-Stokes lines. Suitable methods of population transfer are OARP (Optical Adiabatic Rapid Passage) methods, in particular STIRAP (Stimulated Raman Adiabatic Passage) methods, which are used for example in J. Chem. Phys. 92 (1990), 5363-5376 , are described.

In Fig. 2, the conditions when using the method according to the invention are shown schematically. Irradiated pump light of the mid-infrared spectral range MIR is absorbed by the analyte molecules and causes the number of analyte molecules in the vibratory ground state ν = 1 to decrease relative to the population distribution shown in FIG increases the number of analyte molecules in the first excited vibration state ν = 2. For illustration, therefore, in Fig. 2 on the line of the vibration ground state ν = 1, a much smaller circle than in Fig. 1 is shown. Correspondingly, the circle on the line of the first excited vibration state ν = 2 is correspondingly larger than shown in FIG.

In order to illustrate how the intensity ratios of Stokes and anti-Stokes lines change when the method according to the invention is used, the intensities of a Stokes line S and an associated anti-Stokes line AS are shown in FIG Difference .DELTA.k is plotted between the wavenumber of the primary light and the wavenumber of the respective Raman line S or AS. In this case, the line intensities observed in the prior art are shown in solid lines in the case of thermal occupation of the vibration states according to FIG. 1. Shown are dashed lines the intensity ratios of the Stokes and anti-Stokes lines in accordance with the invention irradiation of pump light of the middle infrared range of FIG. 2.

Fig. 3 makes it clear that the intensity of the anti-Stokes line can be increased drastically by the method according to the invention. Typically, upon irradiation of pump light of the mid-infrared spectral range, the proportion of analyte molecules in the first excited vibration state ν = 2 changes from about 0.8% to almost 50%. This requires in this example an increase in intensity of the anti-Stokes line by a factor of 62.

Since anti-Stokes lines are not affected by fluorescent light, with the method according to the invention a spectroscopic examination of samples consisting of human or animal tissue, body fluids or gases were obtained, with a detection sensitivity possible, which could not even be reached so far with spectroscopic examination methods.

The advantages associated with the irradiation of pump light can not be used for the "classical" Raman spectroscopy, but of course for special Raman spectroscopic methods, such as CARS (Coherent Anti-Stokes Raman Spectroscopy) or SERS (Surface Enhanced Raman Spectroscopy) ,

To further increase the detection sensitivity, the Raman spectrum obtained with irradiation of pump light is preferably used as the comparison Raman spectrum, which is compared with a base Raman spectrum measured on the sample without irradiating pump light. In this way, disturbing influences on the anti-Stokes line to be evaluated can be largely eliminated. Interference, such as ambient light, namely, does not change when irradiated by pump light. Simple subtraction and / or division can therefore computationally compensate disturbing influences on the comparison Raman spectrum with the basis Raman spectrum. If the pump light is switched on and off several times and several comparison and basic Raman spectra are measured successively, interference effects can be better compensated by using statistical methods.

Turning the pump light on and off is a particular form of modulation of the intensity of the pump light. Generally speaking, it is preferable to modulate the intensity of the pump light to give Raman lines based on associated changes in their intensity to identify better. In this case, the intensity of the pump light is particularly preferably modulated with a modulation frequency which is used in the detection of the Raman spectrum in a lock-in method, since lock-in methods are particularly suitable for the elimination of disturbing influences.

In addition to the detection of anti-Stokes lines, Stokes lines of the Raman spectrum are also preferably detected and evaluated. In particular, in complex spectra with a large number of lines, the identification of spectral lines and thus of the corresponding analyte can be facilitated by assigning anti-Stokes lines to the associated Stokes line.

For most organic analytes pump light of the mid-infrared spectral range is most suitable for excitation of vibrational states. Especially in gases, molecules can often be better identified by characteristic rotation bands. In order to excite the corresponding rotational states, pumping light of the far-infrared range is generally best suited. With pump light in the terahertz range (preferably 1 terahertz to 100 terahertz) can vibrational states with small binding constants, such as occur in the connection between the individual strands of a DNA helix, stimulate, so that even such complex organic molecules with the inventive method quantitatively and qualitatively.

4 shows a schematic representation of a Raman spectrometer for carrying out the method according to the invention. The illustrated Raman spectrometer comprises a primary light source 1 for irradiating the sample 2 which is in a cuvette 11, with narrow-band primary light 3 of the near infrared, visible or ultraviolet spectral range, a detector 4 for detecting secondary light 5, which is produced by interaction of the primary light 3 with the sample 2, and an evaluation unit 6 for evaluating a measured Raman spectrum.

For the Raman lines of a Raman spectrum to be reliably identified and evaluated, narrowband primary light 3 must be used. The broader the primary light 3, the greater the line width of the Raman lines and the worse the Raman lines can be separated from the primary light 3, especially when a large line width leads to superimpositions. Therefore, the line width of the irradiated primary light 3 is preferably at most half the distance to the center of the Raman line to be evaluated. For the analytes usually to be determined (for example glucose or cholesterol), this means that the line width of the primary light 3 should correspond to at most 500 cm ^-1 . The line width of a line is understood to be the difference of the wavenumbers in which the spectral light intensity is half of a maximum value reached in the center of the line.

In the exemplary embodiment shown, the primary light source 1 is a laser with which radiation of a wavelength between 2500 nm and 10 nm can be emitted.

By interaction of the primary light 3 with the sample 2 secondary light 5 is detected with a measuring optics 12 and a light guide 13 to a spectral apparatus 7, preferably a grid supplied. After a Spectral decomposition, the spectral intensity distribution of the secondary light 5 with the detector 4, preferably a CCD device measured. The detector 4 is connected to the evaluation unit 6, with which the Raman spectrum obtained in this way is evaluated. With regard to primary light source 1, spectral apparatus 7 and detector 4, the illustrated Raman spectrometer corresponds to the known prior art, so that further explanations are not required for this purpose. Other Raman spectrometer types, in particular Fourier transform Raman spectrometers, can also be used for the method described above.

As a special feature, the Raman spectrometer shown in FIG. 4 has a pump light source 8 for emitting pump light 9 of the middle infrared spectral range. By simultaneously irradiating the sample 2 with the primary light 3 and the pumping light 9, vibration states of the analyte contained in the sample 2 are excited, so that the intensity of the anti-Stokes lines to be evaluated increases drastically.

The primary light 3 can, as in the exemplary embodiment shown, be combined with the pumping light 9 via a beam splitter 14 and supplied to the sample 2 in a superimposed beam. However, the primary light 3 and the pumping light 9 can also be radiated onto the sample 2 at any angle to one another. Likewise, the secondary light 5 can be measured at any angle and in particular also in backscatter.

In the simplest case, broadband medium infrared light sources, for example a globar, can be used as the pump light source 8. However, especially advantageous are Narrow-band pump light sources 8, in particular medium infrared lasers, since in this way targeted vibration states of the analyte molecules of interest can be excited. In this way only anti-Stokes lines of the analyte molecules are amplified. If a broadband pump light source is used, the intensity of the anti-Stokes lines of all molecules contained in the sample increases, which can lead to a slightly higher background. Narrow-band pump light sources can be realized with thermal mid-infrared light sources by respective filters or, preferably, by laser. In addition to lead salt lasers, quantum cascade lasers are particularly suitable.

Preferably, the MIR laser used as the pumping light source 8 is tuned, so that the narrow-band pumping light 9 sweeps over a predetermined frequency range in the Raman spectroscopic examination of the sample 2. In this way, vibrational states of different molecules in the sample are successively excited so that the anti-Stokes lines of different analyte molecules having an increased intensity and low background can be detected one after the other. A further increase in the signal-to-noise ratio can be achieved by using differently polarized primary light 3 and pumping light 9. In Fig. 4 different polarization directions of the primary light 3 and the pump light 9 are indicated by double arrows 15.

FIG. 5 shows a further exemplary embodiment of a Raman spectrometer for carrying out the method according to the invention. Identical and corresponding parts are marked with matching reference numbers.

The essential difference from the exemplary embodiment explained with reference to FIG. 4 is that the primary light 3 is fed via a light guide 17 to a measuring optics 12 which is movable relative to the sample 2. Backscattered secondary light 5 is detected by the measuring optics 12 and, as explained with reference to FIG. 4, fed to the spectral apparatus 7 via a light guide 13.

In this way, with the construction shown in Fig. 5, the sample 2 can also be investigated spatially resolved. Namely, only in the portion 16 of the sample 2 in which intersect the primary light beam 3 and the focused pumping light beam 9, namely secondary light 5 with intensity-increased anti-Stokes lines. By means of a corresponding displacement of the measuring optics 12, it is therefore possible to examine different partial regions of the sample 2 in a spatially resolved manner step by step. By a corresponding mobility of the pumping light source 8, all partial regions of the sample 2 can be examined one after the other.

The spatial resolution can be further improved by the pumping light 9 is directed in each case only to a portion of the sample 2. For this purpose, the pumping light source 8 is correspondingly movable.

Of particular importance in this context is that the spatial resolution of the microspectroscopic method is limited only by the width of the primary light beam 3. Since the primary light beam 3 can be focused in principle on a surface which corresponds approximately to the square of its wavelength, an excellent spatial resolution is possible in this way. As already mentioned, the pumping light 3 is light of the near infrared, visible or ultraviolet spectral range, so that its wavelength is less than 2.5 microns, preferably less than 1 micron.

In this context, it is particularly favorable when the pumping light 9 is fanned out, so that in each case a planar portion of the sample 2 is irradiated by the pumping light 9. In this way, the required for a rasterization of the sample movements of the pumping light source 8 can be reduced to a minimum.

The structure explained with reference to FIG. 5 can be used not only for the examination of biological samples, for example tissue samples, but, with appropriate modifications, for example also for the measurement of trace substances in the air. An actual sample recording is not necessary for many applications. For example, it is possible to cross a pumping light beam with a primary light beam in a volume element of the air to be examined, for example over a chimney, and to evaluate the Raman spectrum of backscattered secondary light, for example to detect pollutants in exhaust gases. The method according to the invention can also be used as a LIDAR method (Light Detection and Ranging), whereby only one pump light source has to be added to the structure customary for LIDAR methods.

With the described method, it is possible to achieve such a high degree of accuracy that, for example, carbon dioxide molecules with ¹³ C isotopes of carbon dioxide molecules can be distinguished from ordinary respirable ¹² C isotopes in the respiratory air emitted by a human. By administering a substance labeled with ¹³ C isotopes to a subject (for example medicaments, carbohydrates, proteins, etc.), it is possible to use the substance according to the invention Procedures are being studied, we will quickly degrade the relevant substances in the body.

LIST OF REFERENCE NUMBERS

1

Primary light source

2

sample

3

primary light

4

detector

5

secondary light

6

evaluation

7

Spectroscopic

8th

Pump light source

9

pump light

11

cuvette

12

measurement optics

13

optical fiber

14

beamsplitter

15

polarization directions

16

Part of the sample

17

optical fiber

S

Stokes line

AS

Anti-Stokes line

ME

Medium infrared light

Claims (24)

Raman spectroscopic analysis method for determining the presence and / or concentration of an analyte in the sample (2), comprising the following steps: Irradiating the sample (2) with narrow band primary light (3) of the near infrared, visible or ultraviolet spectral region, Detecting secondary light (5), which is produced by interaction of the primary light (3) with the sample (2), Recording and evaluating at least one spectral sub-region of the secondary light containing at least one Raman line for determining the presence and / or concentration of an analyte in the sample (2), characterized in that
the specimen (2) is irradiated with pump light (9) of the middle or far infrared spectral range for exciting vibrational and / or rotational states of the analyte contained in the specimen (2), so that when the primary light (3) interacts with the specimen (3) 2) for at least one state of vibration or rotation of the analyte contained in the sample (2), there is an increased occupation number compared to the thermal equilibrium. Method according to Claim 1, characterized in that the at least one recorded and evaluated spectral subarea of the secondary light (5) contains an anti-Stokes line. Method according to claim 1 or 2, characterized in that the sample (2) is irradiated simultaneously with pumping light (9) and primary light (3). Method according to one of the preceding claims, characterized in that; in that the intensity of the pumping light (9) is modulated in order to better associate at least one line in the spectrum of the secondary light (15) with the associated changes in its intensity to the analyte. Method according to Claim 4, characterized in that the intensity of the pumping light (9) is modulated with a modulation frequency which is used in the detection of the secondary light spectrum in a lock-in method. Method according to one of the preceding claims, characterized in that the secondary light spectrum obtained with irradiation of pumping light (9) serves as a comparison spectrum which is compared with a basic secondary light spectrum which is applied to the sample (2) without irradiation of pumped light ( 9) is measured. Method according to Claim 6, characterized in that the pumping light (9) is switched on and off several times and several comparison and base secondary light spectra are measured in succession. Method according to one of the preceding claims, characterized in that a laser is used as the primary light source (1). Method according to one of the preceding claims, characterized in that broadband pumping light (9) is used, which is preferably emitted by a globar. Method according to one of claims 1 to 8, characterized in that narrow-band pumping light (9) is used. Method according to Claim 10, characterized in that the pumping light (9) is emitted by an IR laser, preferably a MIR or FIR laser. Method according to Claim 11, characterized in that the IR laser is a quantum cascade laser. Method according to Claim 10 or 11, characterized in that the frequency of the narrow-band pumping light (9) is tuned during the spectroscopic examination of the sample (2) over a predetermined frequency range. Method according to one of the preceding claims, characterized in that polarized primary light (3) is used. Method according to one of the preceding claims, characterized in that polarized pumping light (9) is used. Method according to one of the preceding claims, characterized in that, for a spatially resolved examination, only a partial region (16) of the sample (2) is irradiated both with primary light (3) and with pump light (9). A method according to claim 16, characterized in that in each case a partial region (16) of the sample (2) is examined in that in each examination step only the respective partial region (16) is illuminated both with primary light (3) and with pump light (9). is irradiated. A method according to claim 16 or 17, characterized in that the primary light (3) is focused on the respectively to be examined portion (16). Method according to one of Claims 16 to 18, characterized in that the pumping light (9) is irradiated onto the sample (2) as a pump light beam which is widened in a plane, so that the primary light (9) strikes a flat partial region of the sample (2). acts. Method according to one of the preceding claims, characterized in that the sample is gaseous, in particular contains breathing gas expelled from a human or animal. Use of a method according to any one of the preceding claims for assaying a sample (2) derived from human or animal tissue or body fluids. Use of a method according to any one of claims 1 to 20 for determining a carbohydrate, lipid or protein concentration. Use of a method according to any one of claims 1 to 20 for determining a glucose, total cholesterol, triglyceride concentration, HD-lipid or LD-lipid concentration. Raman spectrometer for carrying out a method according to one of the preceding claims 1 to 21 with a primary light source (1) for irradiating the sample (2) with narrow-band primary light (3) of the near infrared, visible or ultraviolet spectral range,
a detector (4) for detecting secondary light (5) produced by interaction of the primary light (3) with the sample (2), and
an evaluation unit (6) for evaluating recorded Raman lines of the secondary light (5),
characterized in that
the spectrometer comprises a pump light source (8) for irradiating the sample (2) with pump light (9) of the narrow or far infrared spectral range.

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